P element derived vector and methods for its use

ABSTRACT

Novel P element derived vectors and methods for their use to insert an exogenous nucleic acid into the genome of a target cell are provided. The subject vectors have a pair of P element transposase recognized insertion sites, e.g. 31 base pair inverted repeats, flanking at least two transcriptionally active genes. In practicing the subject methods, a vector of the subject invention is introduced into the target cell under conditions sufficient for transposition to occur. The subject methods find use in a variety of applications in which the insertion of an exogenous nucleic acid into the genome of a target cell is desired, e.g. include research, synthesis and therapeutic applications.

CROSS REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. §119 (e), this application claims priority to thefiling date of the United States Provisional Patent Application Ser. No.60/131,406 filed Apr. 28, 1999; the disclosure of which are hereinincorporated by reference.

INTRODUCTION

1. Field of the Invention

The field of this invention is nucleic acid vectors.

2. Background of the Invention

The introduction of an exogenous nucleic acid sequence (e.g. DNA) into acell, a process known as “transformation,” plays a major role in avariety of biotechnology and related applications, including research,synthetic and therapeutic applications. Research applications in whichtransformation plays a critical role include the production oftransgenic cells and animals. Synthetic applications in whichtransformation plays a critical role include the production of peptidesand proteins. Therapeutic applications in which transformation plays akey role include gene therapy applications. Because of the prevalentrole transformation plays in the above and other applications, a varietyof different transformation protocols have been developed.

In many transformation applications, it is desirable to introduce theexogenous DNA in a manner such that it is incorporated into a targetcell's genome. One means of providing for genome integration is toemploy a vector that is capable of homologous recombination. Techniquesthat rely on homologous recombination can be disadvantageous in that thenecessary homologies may not always exist; the recombination events maybe slow, etc. As such, homologous recombination based protocols are notentirely satisfactory.

Accordingly, alternative viral based transformation protocols have beendeveloped, in which a viral vector is employed to introduce exogenousDNA into a cell and then subsequently integrate the introduced DNA intothe target cell's genome. Viral based vectors finding use includeretroviral vectors, e.g. Maloney murine leukemia viral based vectors.Other viral based vectors that find use include adenovirus derivedvectors, HSV derived vectors, sindbis derived vectors, etc. While viralvectors provide for a number of advantages, their use is not optimal inmany situations. Disadvantages associated with viral based vectorsincluding immunogenicity, viral based complications, and the like.

Accordingly, there is continued interest in the development ofadditional vectors for use in transformation protocols. Of particularinterest is the development of non-viral vectors that provide for stableintegration of exogenous DNA in a cell genome through a mechanism otherthan homologous recombination.

Relevant Literature

U.S. Patents of interest include: U.S. Pat. Nos. 5,719,055 and4,670,388. Other references of interest include: Rio et al.,“Identification and Immunochemical Analysis of Biologically ActiveDrosophila P Element Transposase,” Cell (Jan. 17, 1986) 44:21-32; andRio et al., “Evidence for Drosophila P Element Transposase Activity inMammalian Cells and Yeast,” J. Mol. Biol. (1988) 200: 411-415.

Additional articles of interest include: Schouten et al., Nuc. AcidsRes. (1998) 26:3013-3017; Ivics et al., Cell (1997) 91: 501-510; Luo etal., Proc. Nat'l Acad. Sci USA (1998) 95:10769-10773; and Ivics et al.,Proc. Nat'l Acad. Sci. USA (1996) 93:5008-5013.

SUMMARY OF THE INVENTION

P element derived vectors and methods for their use in the insertion ofan exogenous nucleic acid into a target cell genome are provided. Thevectors of the subject invention include a pair of P element transposaserecognized insertion sequences, e.g. P element derived 31 base pairinverted repeats, flanking at least two transcriptionally active genes.In practicing the subject methods, a vector as described above carryingan exogenous nucleic acid is introduced into a target cell underconditions sufficient for transposition of the exogenous nucleic acidfrom the vector into the target cell genome. The subject methods finduse in a variety of transformation applications, including research,polypeptide synthesis and therapeutic applications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of a vector according to the subject invention.

FIG. 2 provides a diagram showing the synthesis of the vector depictedin FIG. 1.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

P element derived vectors and methods for their use in the insertion ofan exogenous nucleic acid into the genome of a cell are provided. Thesubject vectors include two P element transposase recognized insertionsequences, e.g. P element derived 31 base pair inverted repeats,flanking at least two transcriptionally active genes. In the subjectmethods, a vector according to the invention that includes an exogenousnucleic acid is introduced into a target cell under conditionssufficient for insertion of the exogenous nucleic acid into the targetcell genome to occur. The subject methods find use in a variety ofdifferent applications, including gene therapy applications. In furtherdescribing the subject invention, the subject vectors will be describedfirst, followed by a discussion of the methods of using the subjectvectors for transformation of a target cell.

Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

In this specification and the appended claims, the singular forms “a,”“an,” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

Vectors

The vectors of the subject invention are P element derived vectors, i.e.P element derived transposon vectors, by which is meant that the vectorsinclude at least the P element transposase recognized insertionsequences of the Drosophila P element. As such, the subject vectorsinclude a pair of the 31 base pair inverted repeat domain of the Pelement, or the functional equivalent thereof, i.e. a domain recognizedby the P element encoded transposase. The 31 base pair inverted repeat(i.e. P foot) is disclosed in Beall et al., “Drosophila P-elementtransposase is a novel site-specific endonuclease,” Genes Dev (Aug. 15,1997)11(16):2137-51.

In the subject vectors, the pair of P element encoded transposaserecognized insertion sequences (i.e. P feet) flank at least twotranscriptionally active genes. By at least two is meant two or more,usually no more than five, and more usually no more than four, where thenumber of transcriptionally active genes in the vector is often two orthree. In certain embodiments, the exogenous nucleic acid that isinserted into a target cell genome in the subject methods, described ingreater detail infra, is one of the transcriptionally active genes ofthe vectors. By transcriptionally active gene is meant a coding sequencethat is capable of being expressed under intracellular conditions, e.g.a coding sequence in combination with any requisite expressionregulatory elements that are required for expression in theintracellular environment of the target cell with which the subjectvector is to be employed. As such, the transcriptionally active genes ofthe subject vectors typically include a stretch of nucleotides ordomain, i.e. expression module, that includes a coding sequence ofnucleotides in operational combination, i.e. operably linked, withrequisite trascriptional mediation or regulatory element(s). Requisitetranscriptional mediation elements that may be present in the expressionmodule include promoters, enhancers, termination and polyadenylationsignal elements, splicing signal elements, and the like.

Preferably, the expression module includes transcription regulatoryelements that provide for expression of the gene in a broad host range.A variety of such combinations are known, where specific transcriptionregulatory elements include: SV40 elements, as described in Dijkema etal., EMBO J. (1985) 4:761; transcription regulatory elements derivedfrom the LTR of the Rous sarcoma virus, as described in Gorman et al.,Proc. Nat'l Acad. Sci USA (1982) 79:6777; transcription regulatoryelements derived from the LTR of human cytomegalovirus (CMV), asdescribed in Boshart et al., Cell (1985) 41:521; hsp70 promoters,(Levy-Holtzman, R. and I. Schechter (Biochim. Biophys. Acta (1995) 1263:96-98) Presnail, J. K. and M. A. Hoy, (Exp. Appl. Acarol. (1994) 18:301-308)) and the like.

In many embodiments, at least one of the transcriptionally active genesor expression modules present in the subject vectors is a selectablemarker. A variety of different genes have been employed as selectablemarkers, and the particular gene employed in the subject vectors as aselectable marker is chosen primarily as a matter of convenience. Knownselectable marker genes include: the thimydine kinase gene, thedihydrofolate reductase gene, the xanthine-guanine phosporibosyltransferase gene, CAD, the adenosine deaminase gene, the asparaginesynthetase gene, the antibiotic resistance genes, e.g. tet^(r), amp^(r),Cm^(r) or cat, kan^(r) or neo^(r) (aminoglycoside phosphotransferasegenes), the hygromycin B phosphotransferase gene, and the like.

In addition, the subject vectors typically include at least onerestriction endonuclease recognized site, e.g. restriction site, locatedbetween the P feet which serves as a site for insertion of an exogenousnucleic acid. A variety of restriction sites are known in the art andmay be included into the vector, where such sites include thoserecognized by the following restriction enzymes: HindIII, PstI, SalI,AccI, HincII, XbaI, BamHI, SmaI, XmaI, KpnI, SacI, EcoRI, and the like.In many embodiments, the vector includes a polylinker, i.e. a closelyarranged series or array of sites recognized by a plurality of differentrestriction enzymes, such as those listed above.

The inter P feet domain of the vectors, i.e. that domain or region ofthe vector located or positioned between the P feet which includes theat least two transcriptionally active genes and the exogenous nucleicacid, when present, may vary greatly in size. Typically, the size ofthis inter P feet domain (i.e. P feet flanked domain) is at least about50 bp in length, usually at least about 1000 bp in length and moreusually at least about 2000 bp in length, where the length of thisdomain may be as long as 150,000 bp or longer, but generally does notexceed about 20,000 bp in length and more usually does not exceed about10,000 bp in length.

In certain embodiments, the subject vectors further include atransposase encoding domain, i.e. a region of nucleotides having asequence that encodes a protein having transposase activity,particularly a transposase activity that recognizes the P feet of the Pelement, specifically a protein having the P element transposaseactivity, i.e. P element transposase or a functional equivalent ormimetic thereof. The amino acid sequence of the P element transposase isdisclosed in Rio et al., Cell (Jan. 17, 1986) 44: 21-32. A specifictransposase encoding nucleic acid that may be present on the subjectvectors is that found in pTURBO, where the sequence of this plasmid isdisclosed in W. R. Engels, Bioess. 14: 681-686 (1992). When present onthe subject vectors, this P element transposase encoding region ordomain is located outside the region flanked by the P feet. In otherwords, the transposase encoding region is located external to the regionflanked by the P feet, i.e. outside the inter P-feet domain describedsupra. Put another way, the tranposase encoding region is positioned tothe left of the left terminal P foot or the right of the right terminalP foot.

The subject vectors can be used to stably insert a wide variety ofendogenous and/or exogenous nucleic acids into the genome of a targetcell (exogenous means a nucleic acid having a sequence that is notpresent in the target cell while endogenous means a nucleic acid thatpre-exists in the target cell, prior to insertion). The nature of thenucleic acid will vary depending the particular protocol beingperformed. For example, in research applications, the exogenous orendogenous nucleic acid may be a novel gene whose protein product is notwell characterized. In such applications, the vector is employed tostably introduce the gene into the target cell and observe changes inthe cell phenotype in order to characterize the gene. Alternatively, inprotein synthesis application, the exogenous or endogenous nucleic acidencodes a protein of interest which is to be produced by the cell. Inyet other embodiments where the vector is employed as a gene therapyvector, the exogenous or endogenous nucleic acid is a gene havingtherapeutic activity, i.e. a gene that encodes a product of therapeuticutility. The nucleic acid may vary greatly in size. Generally, the sizeof the nucleic acid that is carried by the vector is at least about 50bp, usually at least about 1000 bp and more usually at least about 2000bp, where the length may be as long as 150,000 bp or longer, butgenerally does not exceed about 20,000 bp and usually does not exceedabout 10,000 bp. In many embodiments, the exogenous nucleic acid islong, by which is meant that it ranges in length from about 30 to 50 kb.

The subject vectors may further comprise one or more elements requiredfor amplification of the vector in a prokaryotic host, e.g. E. coli.Elements that may be included on the vector for use in amplification ofthe vector in a prokaryotic host include: an origin of replication, aselectable marker, and the like.

A representative vector of the subject invention is depicted in FIG. 1.

In addition to the above described vectors that include at least twotranscriptionally active genes, also provided are vectors that include asingle transciptionally active gene. In vectors of this embodiment ofthe subject invention, the promoter that is part of thetranscriptionally active gene may be any of those described above, e.g.SV40, with the proviso that the promoter is not a CMV promoter. Vectorsof this embodiment that include a single transcriptionally active genemay be prepared and used as described below, where the followingdescription is provided in terms of vectors that include at least twotranscriptionally active genes.

Methods of Preparing the Subject Vectors

The vectors of the subject invention may be produced by standard methodsof restriction enzyme cleavage, ligation and molecular cloning. Oneprotocol for constructing the subject vectors includes the followingsteps. First, purified nucleic acid fragments containing desiredcomponent nucleotide sequences as well as extraneous sequences arecleaved with restriction endonucleases from initial sources, e.g theDrosophila P element. Fragments containing the desired nucleotidesequences are then separated from unwanted fragments of different sizeusing conventional separation methods, e.g., by agarose gelelectrophoresis. The desired fragments are excised from the gel andligated together in the appropriate configuration so that a circularnucleic acid or plasmid containing the desired sequences, e.g. sequencescorresponding to the various elements of the subject vectors, asdescribed above is produced. Where desired, the circular molecules soconstructed are then amplified in a prokaryotic host, e.g. E. coli. Theprocedures of cleavage, plasmid construction, cell transformation andplasmid production involved in these steps are well known to one skilledin the art and the enzymes required for restriction and ligation areavailable commercially. (See, for example, R. Wu, Ed., Methods inEnzymology, Vol. 68, Academic Press, N.Y. (1979); T. Maniatis, E. F.Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1982); Catalog1982-83, New England Biolabs, Inc.; Catalog 1982-83, Bethesda ResearchLaboratories, Inc. An example of how to construct a vector of thepresent invention is provided in the Experimental section, infra, anddepicted in FIG. 2.

Methods of Using the Subject Vectors

The subject vectors find use in a variety of applications in which it isdesired to introduce and stably integrate an exogenous or endogenousnucleic acid into the genome of a target cell. As mentioned above,exogenous nucleic acid means a stretch of nucleotides that is notinitially present in the target cell, while endogenous nucleic acidmeans a nucleic acid that pre-exists in the target cell. In manyembodiments, the sequence of nucleotides present in the exogenousnucleic acid will be one that is not found in the genome of the targetcell. The subject methods can be used with a variety of target cells.Target cells with which the subject vectors may be employed aregenerally animal or plant cells, where in many embodiments the targetcells are animal cells. Of particular interest in many embodiments isthe use of the subject vectors to target vertebrate cells, particularlyavian cells, e.g. chicken cells; mammalian cells, including murine,porcine, ovine, equine, rat, dog, cat, monkey, and human cells; and thelike.

In the methods of the subject invention, a P element derived vector asdescribed above is introduced into a target cell under conditionssufficient for excision of the P feet flanked nucleic acid from thevector and subsequent integration of the excised nucleic acid into thegenome of the target cell. As the P element derived vector is introducedinto the cell “under conditions sufficient for excision and integrationto occur,” the subject method further includes a step of ensuring thatthe requisite transposase activity is present in the target cell alongwith the introduced vector. Depending on the structure of the vectoritself, i.e. whether or not the vector includes a region encoding aproduct having P element transposase activity, the method may furtherinclude introducing a second vector into the target cell which encodesthe requisite transposase activity.

The vector (and second vector where necessary) may be introduced intothe target cell using any convenient protocol, where the protocol mayprovide for in vitro or in vivo introduction of the vector. A number ofdifferent in vitro protocols exist for introducing nucleic acids intocells, and may be employed in the subject methods. Suitable protocolsinclude: calcium phosphate mediated transfection; DEAE-dextran mediatedtransfection; polybrene mediated transfection; protoplast fusion, inwhich protoplasts harboring amplified amounts of vector are fused withthe target cell; electroporation, in which a brief high voltage electricpulse is applied to the target cell to render the cell membrane of thetarget cell permeable to the vector; liposome mediated delivery, inwhich liposomes harboring the vector are fused with the target cell;microinjection, in which the vector is injected directly into the cell,as described in Capechhi et al, Cell (1980) 22: 479; and the like. Theabove in vitro protocols are well known in the art and are reviewed ingreater detail in Sambrook, Fritsch & Maniatis, Molecular Cloning: ALaboratory Manual (Cold Spring Harbor Laboratory Press)(1989)pp16.30-16.55. In vivo protocols that find use in delivery of thesubject vectors include delivery via lipid based, e.g. liposomevehicles, where the lipid based vehicle may be targeted to a specificcell type for cell or tissue specific delivery of the vector. Patentsdisclosing such methods include: U.S. Pat. Nos. 5,877,302; 5,840,710;5,830,430; and 5,827,703, the disclosures of which are hereinincorporated by reference. Other in vivo delivery systems may also beemployed, including: the use of polylysine based peptides as carriers,which may or may not be modified with targeting moieties,microinjection, electroporation, and the like. (Brooks, A. I., et al.1998, J. neurosci. Methods V. 80 p: 137-47; Muramatsu, T., Nakamura, A.,and H. M. Park 1998, Int. J. Mol. Med. V. 1 p: 55-62)

The amount of vector nucleic acid that is introduced into the cell issufficient to provide for the desired excision and insertion of theexogenous nucleic acid into the target cell genome. As such, the amountof vector nucleic acid introduced should provide for a sufficient amountof transposase activity and a sufficient copy number of the exogenousnucleic acid. The amount of vector nucleic acid that is introduced intothe target cell varies depending on the efficiency of the particularintroduction or transfection protocol that is employed.

Following introduction of the vector DNA into the target cell incombination with the requisite transposase, the nucleic acid region ofthe vector that is flanked by the P feet of the vector, i.e. the vectornucleic acid positioned between the P element transposase recognized 31base pair terminal repeats, is excised from the vector via the providedtransposase and inserted into the genome of the targeted cell. As such,introduction of the vector DNA into the target cell is followed bysubsequent transposase mediated excision and insertion of the exogenousnucleic acid carried by the vector into the genome of the targeted cell.

Because of the particular properties of the subject transposon basedvectors, the subject vectors may be used to integrate large pieces ofDNA into a target cell genome. Generally, the size of DNA that thevectors insert into a target cell genome is at least about 50 bp,usually at least about 1000 bp and more usually at least about 2000 bp,where the size may be as large as 150,000 bp or larger, but generallydoes not exceed about 20,000 bp and usually does not exceed about 10,000bp. Where the vector inserts a large piece of DNA into a target cellgenome, the size of the inserted DNA ranges from about 30 to 150 kb.

The subject methods of stable integration of exogenous nucleic acid intothe genome of a target cell find use in a variety of applications inwhich the stable integration of an exogenous nucleic acid into a targetcell genome is desired. Applications in which the subject vectors andmethods find use include: research applications, polypeptide synthesisapplications and therapeutic applications. Each of these representativecategories of applications is described separately below in greaterdetail.

Research Applications

Examples of research applications in which the subject vectors find useinclude applications designed to characterize a particular gene. In suchapplications, the vector is employed to insert a gene of interest into atarget cell and the resultant effect of the inserted gene on the cell'sphenotype is observed. In this manner, information about the gene'sactivity and the nature of the product encoded thereby can be deduced.The vectors can also be employed to identify and define DNA sequencesthat control gene expression, e.g. in a temporal (e.g. certaindevelopmental stage) or spatial (e.g. particular cell or tissue type)manner. In such assays, the subject vectors are employed to stablyintegrate into the genome of a target cell a selectable marker gene,e.g. antibiotic resistance, LacZ, etc., where the vector lacks asufficient promoter for the marker the gene such that the marker is notsignificantly expressed, if at all, unless it is underneath anendogenous promoter element. If the marker gene is inserted into thetarget cell genome in sufficient relationship to an endogenous promotersequence, it will be expressed. From the resultant expression profile ofthe marker gene, the endogenous promoter that is mediating itsexpression can then be characterized. Yet another research applicationin which the subject vectors find use is in the identification andcharacterization of the results of gene expression studies. For example,a plurality of distinct vector targeted cells (or animals producedtherefrom) are prepared in which the gene of interest is inserted intodistinct locations in the genome of various targeted cells, whereexpression of the gene of interest is dependent on endogenous promotermediation, i.e. where the gene of interest lacks a promoter or iscoupled to only a weak promoter. By plurality is meant at least two,where the number usually ranges from about 2 to 5000, usually from about2 to 200. This plurality of vector targeted cells may be produced byintroducing the vector in a plurality of cells or taking a collection ofpretargeted cells that are homogenous with respect to the insertion siteof the gene, i.e. progeny of a single targeted cell, and thenintroducing transposase into one or more of, but not all of, theconstituent members of the collection. The subject vectors can also beused to study integration mutants, where a gene of interest is insertedrandomly into the genome and the affects of this random insertion of thetargeted cell phenotype are observed. One can also employ the subjectvectors to produce models in which overexpression and/or misexpressionof a gene of interest is produced in a cell and the effects of thismutant expression pattern are observed. One can also use the subjectvectors to readily clone genes introduced into a host cell viainsertional mutagenesis that yields phenotypes and/or expressionpatterns of interest. In such applications, the subject vectors areemployed to generate insertional mutants through random integration ofDNA. The phenotype and/or expression pattern of the resultant mutant isthen assayed using any convenient protocol. In those mutants ofinterest, cloning of the DNA associated with the phenotype and/orexpression pattern of interest is readily accomplished through use ofthe P-feet of the subject vector.

Polypeptide Synthesis Applications

In addition to the above research applications, the subject vectors alsofind use in the synthesis of polypeptides, e.g. proteins of interest. Insuch applications, a vector that includes a gene encoding thepolypeptide of interest in combination with requisite and/or desiredexpression regulatory sequences, e.g. promoters, etc., (i.e. anexpression module) is introduced into the target cell that is to serveas an expression host for expression of the polypeptide. Followingintroduction and subsequent stable integration into the target cellgenome, the targeted host cell is then maintained under conditionssufficient for expression of the integrated gene. Once the transformedhost expressing the protein is prepared, the protein is then purified toproduce the desired protein comprising composition. Any convenientprotein purification procedures may be employed, where suitable proteinpurification methodologies are described in Guide to ProteinPurification, (Deuthser ed.) (Academic Press, 1990). For example, alysate may be prepared from the expression host expressing the protein,and purified using HPLC, exclusion chromatography, gel electrophoresis,affinity chromatography, and the like.

Therapeutic Applications

The subject vectors also find use in therapeutic applications, in whichthe vectors are employed to stably integrate a therapeutic nucleic acid,e.g. gene, into the genome of a target cell, i.e. gene therapyapplications. The subject vectors may be used to deliver a wide varietyof therapeutic nucleic acids. Therapeutic nucleic acids of interestinclude genes that replace defective genes in the target host cell, suchas those responsible for genetic defect based diseased conditions; geneswhich have therapeutic utility in the treatment of cancer; and the like.Specific therapeutic genes for use in the treatment of genetic defectbased disease conditions include genes encoding the following products:factor VIII, factor IX, β-globin, low-density protein receptor,adenosine deaminase, purine nucleoside phosphorylase, sphingomyelinase,glucocerebrosidase, cystic fibrosis transmembrane regulator,α-antitrypsin, CD-18, ornithine transcarbamylase, arginosuccinatesynthetase, phenylalanine hydroxylase, branched-chain α-ketoaciddehydrogenase, fumarylacetoacetate hydrolase, glucose 6-phosphatase,α-L-fucosidase, β-glucuronidase, α-L-iduronidase, galactose 1-phosphateuridyltransferase, and the like. Cancer therapeutic genes that may bedelivered via the subject vectors include: genes that enhance theantitumor activity of lymphocytes, genes whose expression productenhances the immunogenicity of tumor cells, tumor suppressor genes,toxin genes, suicide genes, multiple-drug resistance genes, antisensesequences, and the like. Because of the length of nucleic acid that canbe carried by the subject vectors, the subject vectors can be used tonot only introduce a therapeutic gene of interest, but also anyexpression regulatory elements, such as promoters, and the like, whichmay be desired so as to obtain the desired temporal and spatialexpression of the therapeutic gene.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

I. Preparation of a P Element Derived Vector

The Gene Transfer Vector depicted in FIG. 1 was prepared by digestingthe pcdna3.1hislacz vector shown in FIG. 2 with the restrictionendonucleases BglII and BstBI. This gave rise to a 6 kb fragment and a2.5 kb fragment of DNA. The 6.5 kb fragment contained the neo and laczgenes and was used to construct the gene transfer vector. pCasper4(depicted in FIG. 2) was treated with restriction endonucleases ClaI andBamHI. This gave rise to a 2.8 kb and a 5 kb fragment of DNA. The 5 kbfragment that contained the P element inverted repeats, which arerequired for P element mobilization and integration, was used toconstruct the gene transfer vector. The 5 kb ClaI, BamHI fragment of DNAfrom pCasper4 was ligated to the 6 kb BglII, BstBI fragment of DNA frompcdna3.1hislacz. This formed the new vector as diagramed below. Variantsof this vector have been made that continue to function. Variants thathave been made include replacing the lacz gene with the gfp gene andadding a multiple cloning polylinker.

II. Transfection of Cells with a P Element Derived Vector

The ability of the vector produced in I above to transfect a number ofdifferent types of cells was tested.

A. QT6 Fibroblast Cells

A P element derived vector as produced in 1 above was transientlytransfected into cultured QT6 fibroblast cells. A separate vector,pTURBO (W. R. Engels, Bioess. 14: 681-686 (1992) that contained the Pelement transposase gene was also transfected into these cells. Theintegration of the P element transposon domain of the vector (i.e. thatdomain of the vector flanked by the P feet) was determined by long termgrowth (13 days) on medium containing 200 μg/ml of G418, a neomycin likeantibiotic. The G418 resistant cells were all betagalactosidase positive(not shown). G418 resistance was dependent on both the P element basedvector and the P transposase vector being co-transfected into the QT6cells. Either vector alone yielded no G418 resistant cells. In addition,the number of G418 resistant cells is concentration dependent upon theamount of DNA introduced into the cell. The results are provided inTable 1, infra. When 1 μg of the P element w/lacZ and G418r genes wastransfected into QT6 cells alone, lacz gene expression was no longerdetected in cells not under G418 treatment after 10 days (not shown).Transfections were carried out using standard calcium chlorideprotocols.

TABLE 1 The P element based vector containing the G418r gene confersstable resistance only when introduced into QT6 cells along with thetransposase producing vector. DNA transfected into cells (μg) % of cellssurviving 13 days Transposase P element based vector under 200 μg/ml ofG418 producing vector w/lacZ + G418^(r) selection* 0 0 0 0 1 0 3 0 0 .1.5 1-5%  .5 .5 20% 3 .5 50% .5 .5 20% .5 2 25% .5 5 50% *Percentage isdetermined by the number of cells that survive/total number of originalcells, taking into account doubling time.

QT6 cells transfected with transposase and P element maintain the geneexpression for both the lacZ and G418r for at least 30 days.

The above results indicate that the P element based transposon vectorintegrates into the genome in a transposase dependent manner.

To determine the vector attributes necessary for transformation of QT6cells, as described above, various control vectors were assembledaccording to the methods described above and the ability of thesecontrols to transfect QT6 cells was determined.

The results are provided in Table 2.

TABLE 2 DNA transfected into cells % of cells (μg) surviving 13 daysTransposase P element P element based under 200 μg/ml producing basedvector vector w/G418^(r) + of G4l8 vector w/G418^(r) only BluescriptDNA* selection* .1 .5 — 0 .5 .5 — 0 3 .5 — 0 3 1 — 0 .5 2 — 0 .5 5 — 0.1 — .5 0 .5 — .5 0 3 — .5 0 3 — 1 0 .5 — 2 0 .5 — 5 0 *Percentage isdetermined by the number of cells that survive/total number of originalcells, taking into account doubling time.

The above results indicate that successful integration and geneexpression is dependent upon having two transcriptionally active genes.One gene alone (G418^(r)) does not enable integration. In addition, thesize of the vector is not an important requirement as atranscriptionally inert DNA was substituted in place of the lacZ gene,which kept the overall size of the construct constant.

B. Mouse Embryonic Stem Cells (CCE)

The vector prepared in I above was used to integrate the kanamycin andbetagalactosidase genes into mouse embryonic stem cells in a methodanalogous to that described in IIA above. The results are provided inTable 3.

TABLE 3 DNA transfected into cells (μg) Transposase P element based % ofcells surviving 20 days producing vector w/G418^(r) under 200 μg/ml ofG418 vector and LacZ selection* 0 0 0 1 0 0 0 1 0 1 1 50%

D. Human Kidney Cells (293)

The vector prepared in I above was used to integrate the kanamycin andbetagalactosidase genes into human kidney cells in a method analogous tothat described in IIA above. The results are provided in Table 4.

TABLE 4 DNA transfected into cells (μg) Transposase P element based % ofcells surviving 13 days producing vector w/G418^(r) under 400 μg/ml ofG418 vector and LacZ selection* 0 0 1% 1 0 1% 0 1 5% 1 1 70% 

It was difficult to find an antibiotic treatment which would kill allcells that were being analyzed.

The above results demonstrate that the subject P element based vectorsare capable of stably integrating exogenous nucleic acids into thegenome of vertebrate cells, and in particular mammalian cells. As such,the above results demonstrate that the subject vectors are suitable foruse as vectors for use in the introduction of exogenous nucleic acidsinto mammalian cells.

E. Integration of a Large DNA Fragments into a Target Cell

P element vectors have the capability to mobilize up to 150 kb of DNA .This presents the opportunity of the P element carrying a DNA sequencewith therapeutic use of similar lengeth into a target cell. Themanipulation and handling of DNA fragments up to 4 megabases isdifficult but has been reproducibly achieved. For example, this vectorcould allow the potential for analyzing genes on the size of the humanhuntington gene (˜150-200 kb).

III. Gene Therapy

Once the gene transfer vector has been engineered with the desired geneand its regulatory sequences according to the methods described above,the construct must reach the receipient cell's nucleus to achieve geneintegration. Many methods exist to achieve the transfer of this vectorinto a recipient cell. For instance microinjection into mouseoocyte/embryo nuclei would be optimal for creating a germ line transferof genetic material. This could also be achieved by transfecting mouseembryonic stem cells, selecting the cells that have had the desiredgenetic material transferred into the genome and then implanting thesecells into pseudo-pregnant females.

Several methods have been shown to transfer genetic material into humancells. Direct injection of naked DNA has been shown to be taken up andexpressed transiently in muscle cells. While other methods use lipid orprotein conjugates to facilitate the uptake of DNA. Finally, electriccurrent has also been shown to achieve transfer of DNA in organisms.

VI. Gene Therapy in Mice

A 6 week old male mouse (30 grams in weight) was injected with 5micrograms of the integration vector and 2 micrograms of the transposasecontaining vector. The standard procedure from mirus transit in vivogene delivery system was used for the injection. Briefly, this procedurecombines the DNA to a lipid carrier to help protect the DNA fromdegradation in the blood and to assist the DNA to be uptaken by cells inthe animal. The injection was into the tail vein of the animal. At 15weeks of age, tissue samples were taken from the liver, testis, andtail. Genomic DNA was isolated from these samples using the standardproteinase k/spooling technique. These preparations of genomic DNAs weresubjected to PCR analyses.

The presence of the integration vector was examined using primers to thebeta-galactosidase gene while the presence of the transposase containingvector was examined using primers to the transposase gene. Genomic DNAfrom the tail of the untreated control mouse showed an absence of boththe beta-galactosidase and transposase genes. This control was notsurprising as neither the transposase nor beta-galactosidase genes areendogenous to the mouse genome. However, 9 weeks after the integrationand transposase vectors were injected into the test mouse, thebeta-galactosidase gene was easily detectable. This indicated that theintegration vector had inserted into the genomic DNA in all the tissuesexamined. The absence of the transposase vector in the genomic DNAindicates that DNA did not randomly integrate into the mouse genome andthat the integration vector did so via its standard mechanism. To ensurethat both sets of PCR primers were capable of amplifying a lowconcentration of target DNA, both the transposase and beta-galactosidasegenes are detectable from mixing 0.01 nanograms of integration vectorand transposase vector DNAs into the genomic DNA of the untreated mouse.

TABLE 1 Gene integration in mice PCR results from betagalactosidase PCRresults from DNA gene transposase gene Untreated Tail genomic − − mouseVector Tail genomic + − treated mouse Liver genomic + − Testis genomic +− Untreated Tail genomic + .01 + − mouse ng of betagalactosidase vectorUntreated Tail genomic + .01 − + mouse ng of transposase vector

This data indicates that this vector can be used for somatic cell genetherapy. Furthermore, the integration into testis cells also indicatesthat the subject vector finds use in germ line applications.

It is evident from the above results and discussion that the subjectinvention provides a novel nucleic acid vector having a broad range ofapplications. Advantages of the subject vector over other known nucleicacid vectors include the ability to integrate long stretches of nucleicacid into the genome of a target cell. This feature is advantageous fora number of reasons, including the ability to integrate an exogenousgene along with its native expression regulatory elements. Anotherfeature of the subject invention is that the vectors provide for randominsertion of a foreign nucleic acid, which is desirable in manyapplications. Yet another advantage of the subject invention is that thesubject vectors do not elicit host immune reactions, in contrast withmany viral based vectors. In addition, the subject vectors do not carryrisk of viral infection or recombination hazards. As such, the subjectvectors provide a number of advantages over other vectors currentlyknown and employed in the art, and therefore represent a significantcontribution to the art.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

What is claimed is:
 1. A P element vector for introducing an exogenousnucleic acid into a target non-insect cell genome, said vectorcomprising: a pair of P element transposase recognized insertionsequences flanking at least two non-insect cell transcriptionally activeexpression modules each comprising a coding sequence and a promoter. 2.The vector according to claim 1, wherein said vector further comprisesat least one endonuclease cleavage site.
 3. The vector according toclaim 1, wherein said endonuclease cleavage site is present in apolylinker.
 4. The vector according to claim 1, wherein said vectorfurther comprises transposase domain encoding a product having P elementtransposase activity, wherein said transposase domain is not flanked bysaid pair of transposase recognized insertion sequences.
 5. The vectoraccording to claim 1, wherein said vector further comprises an exogenoussequence positioned at a site between said pair of transposaserecognized insertion sequences.
 6. The vector according to claim 1,wherein said transposase recognized insertion sequences are 31 base pairinverted repeats.
 7. A P element vector for introducing an exogenousnucleic acid into a non-insect target cell genome, said vectorcomprising: a pair of P element derived 31 base pair inverted repeatsflanking at least two non-insect cell transcriptionally active genes,wherein each of said transcriptionally active-expression modules eachcomprising a coding sequence that is expressed under intracellularconditions.
 8. The vector according to claim 7, wherein said vectorfurther comprises a nucleic acid sequence encoding a product having Pelement transposase activity positioned external to the vector domainflanked by said pair of P element derived 31 base pair inverted repeats.9. The vector according to claim 7, wherein said vector furthercomprises an exogenous nucleic acid positioned between said P elementderived 31 base pair inverted repeats.
 10. A method of inserting anexogenous nucleic acid into a non-insect target cell genome, said methodcomprising: introducing into said cell a P-element derived vectorcomprising said exogenous nucleic acid under conditions sufficient fortransposition to occur; whereby said exogenous nucleic acid is insertedinto said genome; with the proviso that said target cell is not aninsect cell.
 11. The method according to claim 10, wherein said targetcell is an animal or plant cell.
 12. A method of inserting an exogenousnucleic acid into a non-insect target cell genome, said methodcomprising: introducing into said cell a vector according to claim 1under conditions sufficient for transposition to occur; whereby saidexogenous nucleic acid is inserted into said genome.
 13. The methodaccording to claim 12, wherein said vector comprises a transposasedomain.
 14. The method according to claim 12, wherein said methodfurther comprises introducing a second vector comprising a transposasedomain into said cell.
 15. The method according to claim 12, whereinsaid exogenous nucleic acid ranges in length from about 50 to 150,000bp.
 16. The method according to claim 12, wherein said target cell is avertebrate cell.
 17. The method according to claim 16, wherein saidvertebrate cell is a mammalian cell.
 18. A kit for use in inserting anexogenous nucleic acid into a non-insect target cell, said kitcomprising: a P element vector comprising a pair of P elementtransposase recognized insertion sequences flanking at least twonon-insect cell transcriptionally active expression modules eachcomprising a coding sequence and a promoter.
 19. The kit according toclaim 18, wherein said vector further comprises at least oneendonuclease cleavage site positioned between said transposaserecognized insertion sequences.
 20. The kit according to claim 19,wherein said endonuclease cleavage site is present in a polylinker. 21.The kit according to claim 18, wherein said kit further comprises anucleic acid sequence encoding a product having P element transposaseactivity.
 22. The kit according to claim 21, wherein said vectorcomprises said nucleic acid sequence encoding a product havingtransposase activity.
 23. The kit according to claim 21, wherein saidnucleic acid sequence encoding a product having transposase activity ispresent on a second vector.
 24. The kit according to claim 18, whereinsaid transposase recognized insertion sequences are 31 base pairinverted repeats.